Deposition-related sites K5/K12 in histone H4 are not required for nucleosome deposition in yeast

Abstract

Histone H4 can be acetylated at N-terminal lysines K5, K8, K12, and K16, but newly synthesized H4 is diacetylated at K5͞K12 in diverse organisms. This pattern is widely thought to be important for histone deposition onto replicating DNA. To investigate the importance of K5͞K12 we have mutagenized these lysines in yeast and assayed for nucleosome assembly. Assaying was done in the absence of the histone H3 N terminus, which has functions redundant with those of H4 in histone deposition. Nucleosome assembly was assayed by three methods. Because nucleosome depletion may be lethal, we examined cell viability. We also analyzed nucleosome assembly in vivo and in vitro by examining plasmid superhelicity density in whole cells and supercoiling in yeast cell extracts. All three approaches demonstrate that mutagenizing K5 and K12 together does not prevent cell growth and histone deposition in vivo or in vitro. Therefore, K5͞K12 cannot be required for nucleosome assembly in yeast. It is only when the first three sites of acetylation-K5, K8, and K12-are mutagenized simultaneously that lethality occurs and assembly is most strongly decreased both in vivo and in vitro. These data argue for the redundancy of sites K5, K8, and K12 in the deposition of yeast histone H4.Nucleosome assembly involves the deposition of a tetramer of histones H3 and H4 onto DNA, followed by the association of two histone H2A͞H2B dimers. In this process, acetylation of histone H4 is likely to play a key role. Newly synthesized histone H4 was shown by pulse-labeling to be diacetylated at lysine residues 5 and 12 (K5͞K12), a conserved feature in Tetrahymena, flies, and humans (1-3). This distinct nonrandom pattern of acetylation among the four acetylatable lysines (K5, K8, K12, and K16) has led to the suggestion that K5͞K12 diacetylation serves a unique role in targeting newly synthesized histone H4 for assembly (4). Another argument for the importance of H4 acetylation in nucleosome assembly derives from the study of the human multiprotein complex (CAF-1, for chromatin assembly factor) that enables H3 and H4 assembly onto replicating DNA in a simian virus 40-based cell-free system (5-7). CAF-1 deposits newly synthesized histones but not those extracted from bulk chromatin onto DNA (7), a result which is consistent with the finding that CAF-1 associates preferentially with histone H4 acetylated in a specific manner (8). However, H4 associated with CAF-1 is not uniquely diacetylated at K5 and K12 but is heterogeneously acetylated at K5, K8, and K12. Moreover, some 33% of H4 in the complex is not acetylated. In addition, much of the H3 in the complex is monoacetylated, whereas some 60% of H3 is unacetylated (9).The yeast (Saccharomyces cerevisiae) histone H4 N-terminal sequence and the location of its acetylated lysines are extremely conserved in evolution. While it is not known whether newly synthesized yeast H4 is diacetylated or whether acetylated H4 is associated with a yeast chromatin assembly factor, we set out to ask whether K5͞K12 is requi...

The Histone Chaperone Anti-silencing Function 1 Stimulates the Acetylation of Newly Synthesized Histone H3 in S-phase

“…1B). Although it is possible that the inviability is because of other roles of Asf1 in the cell, this result suggests that loss of H3 lysine 9 and lysine 56 acetylation together with loss of the acetylatable lysines on histone H4 may result in a level of acetylation on newly synthesized histones that falls below the threshold required for chromatin assembly in yeast (14).…”

“…It is currently unknown whether the acetylation marks in the N-terminal tails of the newly synthesized histone H3 are removed rapidly following chromatin assembly, as is the case for H4. The available evidence indicates that acetylation of histone H4 lysines at positions 5 and 12 is not essential for chromatin assembly, although acetylation of either lysines 5, 8, or 12 seems to be required for chromatin assembly in yeast when the N-terminal tail of histone H3 is also deleted (14). Despite its requirement for chromatin assembly, the exact purpose of the acetylation of the N-terminal tails of the newly synthesized histones is unclear.…”

NAD+-dependent deacetylation of H4 lysine 16 by class III HDACs

“…For instance, in yeast, while abrogation of the entire H4 tail had strong defects in histone deposition during DNA replication, mutation of K5 or K12 did not seem to affect deposition and showed strong redundancy with acetylated K8 (Ma et al, 1998). A possible explanation might lie in the observation that acetylation of H4 is important for nuclear import.…”

High-resolution mapping of H4K16 and H3K23 acetylation reveals conserved and unique distribution patterns inArabidopsisand rice

“…25 For example, acetylation at K5, K8, and K12 of H4 are redundant with each other for nucleosome assembly and transcription. 26 Only the combined mutations in H4 K5, K8, and K12 residues exhibit accumulative impacts on gene expression levels, whereas the individual mutation has a negligible effect on transcription. 27 In contrast, acetylation of lysine 16 of histone H4 (H4K16ac) appears to have unique and distinct functions from other acetylation marks.…”